We are pursuing three complementary projects to elucidate the molecular pharmacology of clinically relevant inhibitors of topoisomerases, DNA repair and cell cycle checkpoint inhibitors. Project #1. Repair of topoisomerase cleavage complexes (TOPccs) by tyrosyl-DNA-phosphodiesterases (TDPs), endonucleases and SUMOylation/ubiquitylation Aim 1:Post-translational modifications of TOPccs: TOPccs are excised by two main mechanisms: 1/ hydrolysis of the covalent linkage between the catalytic tyrosine of topoisomerases and the DNA broken end by tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2); 2/ endonuclease cleavage of the DNA fragment adjacent to the TOP1cc by nucleases (Mre11, XPF-ERCC1, XPG...). Because the covalent topoisomerase-tyrosyl-DNA bonds to be cleaved by TDP1 and TDP2 are deep within the TOPccs, TOPccs need to be proteolyzed and/or denatured to provide access to the TDPs (TDP1 and TDP2). We are studying the proteolytic pathways for TOPccs. Our results demonstrate the rapid engagement of the SUMOylation and ubiquitylation pathways, which, in turn drive proteasome-mediated topoisomerase degradation. Aim 2: Biology of TDPs: TDP1 and TDP2 preferentially repair TOP1cc and TOP2cc, respectively. In addition to TOP1cc, TDP1 removes damaged and non-canonical bases and adducts from 3'-DNA ends. This explains why lack of TDP1 sensitizes cells not only to TOP1 inhibitors but also to temozolomide, cytarabine, zidovudine (AZT) and acyclovir. We are studying how TDP1 is regulated and recruited to DNA damaged sites. We previously reported that TDP1 is coupled with PARP1 and that inhibiting PARP1 results in TDP1 inactivation. Also we previously established that TDP1 excises TOP1cc and TOP1MTcc both in the nuclear and mitochondrial genomes, respectively. This year, we demonstrated that TDP2 also removes TOP2cc in the mitochondrial genome. We also showed for the first time that the arginine methyltransferase (PRMT5) activates TDP1 by directly binding and methylating TDP1. Aim 3: Pharmacology and targeting of TDPs: The rationale for targeting TDPs is rooted in the emerging importance of TDPs for DNA repair and viral replication, and the potential of TDP inhibitors for anticancer drug combinations. To do so, we are using biochemical assays with recombinant TDP enzymes. We are also taking advantage of TDP1 and TDP2 knockout cell lines, crystallographic determinations and molecular modeling to study the molecular pharmacology of the drug candidates. Project #2. PARP trapping by PARP inhibitors: molecular mechanisms and translational implications PARP inhibitors represent the most advanced cancer therapeutics targeting the DNA damage response. Four inhibitors (olaparib, rucaparib, niraparib and talazoparib) have been approved recently. PARP inhibitors are the first drugs to exploit the concept of synthetic lethality for homologous recombination deficiency (HRD) in the clinic. Our studies revealed 'PARP trapping' as a key mechanism explaining the molecular mechanism of action of PARP inhibitors as anticancer agents. This discovery and our work with talazoparib contributed to the approval of talazoparib for breast and ovarian cancer in 2018. Our studies focus on the differences in the molecular mechanisms of action between PARP inhibitors with respect to PARP trapping and what this means for both monotherapy activity and combination with chemotherapeutic agents. We are investigating in preclinical models the two most synergistic combinations: with temozolomide and with TOP1 inhibitors, including our non-camptothecin indenoisoquinoline TOP1 inhibitors (see above). Project #3. Genomic determinants of drug response and preclinical models for predictive biomarkers for patient selection and rational drug combinations with TOP1 and PARP inhibitors The current lack of predictive biomarkers for widely used DNA-targeted anticancer therapies and the lack of direct correlation between their primary targets and cellular response warrant the need to identify novel DNA damage response (DDR) determinants for predicting drug responses and rationalizing drug combinations. Aim 1: Use and value of cancer cell line databases to mine drug responses based on CellMiner: Taking advantage of the extensive NCI-60 drug database ( 40,000 drugs including FDA approved and investigational clinical drugs), whole genomic data and our CellMiner facility, we discovered several novel predictive biomarkers for DNA-targeted agents: SLX4 (FANCP) mutations, ATAD5 (ELG1) mutations, and SLFN11 (Schlafen 11) expression. We have now extended these analyses to tissue-specific cancer cell line databases (NCI Small Cell Lung Cancers), and larger databases (CCLE: MIT-Broad Institute and CGP: MGH-Sanger), and to CCR clinical trials to test predictive biomarker signatures. Those cancer cell line databases have been made widely and freely available to the research community via CellMiner web-based application (http://discover.nci.nih.gov/cellminercdb). Aim 2: SLFN11 as predictive biomarkers for response to DNA damaging drugs was discovered through NCI-60 analyses and in parallel by the CCLE teams. SLFN11 determines response to TOP1, TOP2, PARP inhibitors, DNA synthesis inhibitors and platinum derivatives but not to tubulin or protein kinase inhibitors or apoptosis-inducing drugs. SLFN11 is inactivated in approximately 50% of cancer cells lines, making them resistant to DNA damaging agents. Our aims are to elucidate the molecular mechanism of SLFN11 action and regulation, and relevance for patient responses and rationale drug combinations. We discovered a key molecular mechanism of action of SLFN11 by demonstrating that SLFN11 is recruited to DNA damage sites and to stressed replication forks by binding to RPA and the replicative CMG complex. In doing so, SLFN11 blocks elongating replicons and irreversibly blocks replication. We propose that SLFN11 acts as a Restriction Factor for cells with replicative stress. We have also demonstrated that SLFN11 is inactivated epigenetically in approximately 50% of all cancer cell lines and in patient tumors, and that treatment with histone deacetylase (HDAC) inhibitors reactivates SLFN11 expression and overcomes resistance to DNA-targeted anticancer drugs.